杜鹃素通过降低Cx43的表达抑制AngⅡ诱导的VSMCs增殖
|
摘要
目的研究Cx43在杜鹃素抑制血管紧张素Ⅱ(AngⅡ)诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖中的作用。方法体外培养原代大鼠VSMCs细胞,随机分为对照组、AngⅡ刺激组(模型组)、AngⅡ+杜鹃素组(给药组)。CCK-8法检测细胞活力,Ed U法检测细胞增殖活性,流式细胞术观察细胞周期,real-time PCR和Western blot法检测细胞中Cx43的表达。用si RNA干扰Cx43的表达并测定细胞Ed U结合率和细胞周期。结果浓度为60μmol·L~(-1)的杜鹃素可使AngⅡ诱导的大鼠VSMCs的细胞增殖活性和Ed U结合率均明显降低(P<0.05),并阻止VSMCs由G0/G1期向S期的转化。Real-time PCR和Western blot结果显示,与模型组比较,杜鹃素能明显降低Cx43的m RNA和蛋白表达水平(P<0.01)。用si RNA干扰Cx43的表达后,杜鹃素对AngⅡ诱导的VSMCs增殖的抑制作用明显减弱。结论杜鹃素可明显抑制AngⅡ诱导的VSMCs增殖,抑制VSMCs有丝分裂,使其停滞于G_0/G_1期,其作用可能与杜鹃素抑制Cx43的表达有关。
Aim To study the role of Cx43 in inhibition of Ang II-induced vascular smooth muscle cells( VSMCs) proliferation by farrerol. Methods The primary VSMCs were isolated and cultured by direct adherent culture methods. VSMCs were identified by immunohistochemstry. The cells were divided into the following groups: control group,Ang II group,Ang II +Farrerol group. The cell viability was measured by CCK-8 cell vitality test. The proliferation of VSMCs was measured by the methods of Edu. The cell cycle of VSMCs was detected by flow cytometry. The m RNA levels of Cx43 were measured by Real-time PCR. The protein levels of Cx43 were measured by Western blot.Results 60 μmol·L~(-1) farrerol could significantly decrease the cell viability and Ed U rate of VSMCs induced by Ang II( P < 0. 05),which could also prevent the transformation of VSMCs from G_0/G_1 phase to S phase. The results of real-time PCR and Western blot showed that,compared with the model group,Farrerol could significantly reduce the m RNA and protein expression level of Cx43( P < 0. 01). After the interference of Cx43 by si RNA,the inhibition of proliferation by farrerol decreased significantly. Conclusion Farrerol inhibits Ang II-induced VSMCs proliferation significantly,which might be associated with reducing the expression of Cx43.
Aim To study the role of Cx43 in inhibition of Ang II-induced vascular smooth muscle cells( VSMCs) proliferation by farrerol. Methods The primary VSMCs were isolated and cultured by direct adherent culture methods. VSMCs were identified by immunohistochemstry. The cells were divided into the following groups: control group,Ang II group,Ang II +Farrerol group. The cell viability was measured by CCK-8 cell vitality test. The proliferation of VSMCs was measured by the methods of Edu. The cell cycle of VSMCs was detected by flow cytometry. The m RNA levels of Cx43 were measured by Real-time PCR. The protein levels of Cx43 were measured by Western blot.Results 60 μmol·L~(-1) farrerol could significantly decrease the cell viability and Ed U rate of VSMCs induced by Ang II( P < 0. 05),which could also prevent the transformation of VSMCs from G_0/G_1 phase to S phase. The results of real-time PCR and Western blot showed that,compared with the model group,Farrerol could significantly reduce the m RNA and protein expression level of Cx43( P < 0. 01). After the interference of Cx43 by si RNA,the inhibition of proliferation by farrerol decreased significantly. Conclusion Farrerol inhibits Ang II-induced VSMCs proliferation significantly,which might be associated with reducing the expression of Cx43.
引文
[1]Uryga A K,Bennett M R.Ageing induced vascular smooth muscle cell senescence in atherosclerosis[J].J Physiol,2016,594(8):2115-24.
[2]Chistiakov D A,Orekhov A N,Bobryshev Y V.Vascular smooth muscle cell in atherosclerosis[J].Acta Physiol(Oxf),2015,214(1):33-50.
[3]Bennett M R,Sinha S,Owens G K.Vascular smooth muscle cells in atherosclerosis[J].Circ Res,2016,118(4):692-702.
[4]刘恩荔.杜鹃素新结构衍生物djs-f抑制大鼠血管内膜增生作用及其机制[D].太原:山西医科大学,2016.[4]Liu E L.The inhibitory effect and action mechanism of DJS-F,a new structure derivative of farrerol,on balloon-induced intima hyperplasia in rat common carotid artery[D].Taiyuan:Shanxi Medical University,2016.
[5]Johnstone S R,Ross J,Rizzo M J,et al.Oxidized phospholipid species promote in vivo differential cx43 phosphorylation and vascular smooth muscle cell proliferation[J].Am J Pathol,2009,175(2):916-24.
[6]Jia G,Cheng G,Gangahar D M,et al.Involvement of connexin43 in angiotensinⅡ-induced migration and proliferation of saphenous vein smooth muscle cells via the MAPK-AP-1 signaling pathway[J].J Mol Cell Cardiol,2008,44(5):882-90.
[7]Alonso F,Krattinger N,Mazzolai L,et al.An angiotensinⅡ-and NF-κB-dependent mechanism increases connexin 43 in murine arteries targeted by renin-dependent hypertension[J].Cardiovasc Res,2010,87(1):166-76.
[8]Morel S.Multiple roles of connexins in atherosclerosis-and restenosis-induced vascular remodelling[J].J Vasc Res,2014,51(2):149-61.
[9]王光宇,毕亚光,刘向东,等.二甲双胍对高糖培养H9c2细胞connexin43表达的影响[J].中国药理学通报,2016,32(7):920-4.[9]Wang G Y,Bi Y G,Liu X D,et al.Effect of metformin on connexin43 expression in H9c2 cells cultured with high glucose[J].Chin Pharmacol Bull,2016,32(7):920-4.
[10]Matsushita T,Rama A,Charolidi N,et al.Relationship of connexin43 expression to phenotypic modulation in cultured human aortic smooth muscle cells[J].Eur J Cell Biol,2007,86(10):617-28.
[11]Johnstone S R,Kroncke B M,Straub A C,et al.MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation[J].Circ Res,2012,111(2):201-11.
[12]Song M,Yu X,Cui X,et al.Blockade of connexin 43 hemichannels reduces neointima formation after vascular injury by inhibiting proliferation and phenotypic modulation of smooth muscle cells[J].Exp Biol Med(Maywood,NJ),2009,234(10):1192-200.
[13]Kwak B R,Veillard N,Pelli G,et al.Reduced connexin43 expression inhibits atherosclerotic lesion formation in low-density lipoprotein receptor-deficient mice[J].Circulation,2003,107(7):1033-9.
[14]Shi Y,Hou X,Zhang X,et al.Inhibition of oxidized-phospholipid-induced vascular smooth muscle cell proliferation by resveratrol is associated with reducing Cx43 phosphorylation[J].J Agric Food Chem,2013,61(44):10534-41.
[15]侯晓敏,秦小江.槲皮素通过激活kv1.5保护糖尿病大鼠冠脉损伤[J].中国药理学通报,2017,33(10):1442-5.[15]Hou X M,Qin X J.Quercetin protects coronary artery from injury induced by diabetes in rats by activating Kvl.5[J].Chin Pharmacol Bull,2017,33(10):1442-5.
[16]Liu Y,Fu Y Q,Peng W J,et al.Rutaecarpine reverses the altered connexin expression pattern induced by oxidized low-density lipoprotein in monocytes[J].J Cardiovasc Pharmacol,2016,67(6):519-25.
[2]Chistiakov D A,Orekhov A N,Bobryshev Y V.Vascular smooth muscle cell in atherosclerosis[J].Acta Physiol(Oxf),2015,214(1):33-50.
[3]Bennett M R,Sinha S,Owens G K.Vascular smooth muscle cells in atherosclerosis[J].Circ Res,2016,118(4):692-702.
[4]刘恩荔.杜鹃素新结构衍生物djs-f抑制大鼠血管内膜增生作用及其机制[D].太原:山西医科大学,2016.[4]Liu E L.The inhibitory effect and action mechanism of DJS-F,a new structure derivative of farrerol,on balloon-induced intima hyperplasia in rat common carotid artery[D].Taiyuan:Shanxi Medical University,2016.
[5]Johnstone S R,Ross J,Rizzo M J,et al.Oxidized phospholipid species promote in vivo differential cx43 phosphorylation and vascular smooth muscle cell proliferation[J].Am J Pathol,2009,175(2):916-24.
[6]Jia G,Cheng G,Gangahar D M,et al.Involvement of connexin43 in angiotensinⅡ-induced migration and proliferation of saphenous vein smooth muscle cells via the MAPK-AP-1 signaling pathway[J].J Mol Cell Cardiol,2008,44(5):882-90.
[7]Alonso F,Krattinger N,Mazzolai L,et al.An angiotensinⅡ-and NF-κB-dependent mechanism increases connexin 43 in murine arteries targeted by renin-dependent hypertension[J].Cardiovasc Res,2010,87(1):166-76.
[8]Morel S.Multiple roles of connexins in atherosclerosis-and restenosis-induced vascular remodelling[J].J Vasc Res,2014,51(2):149-61.
[9]王光宇,毕亚光,刘向东,等.二甲双胍对高糖培养H9c2细胞connexin43表达的影响[J].中国药理学通报,2016,32(7):920-4.[9]Wang G Y,Bi Y G,Liu X D,et al.Effect of metformin on connexin43 expression in H9c2 cells cultured with high glucose[J].Chin Pharmacol Bull,2016,32(7):920-4.
[10]Matsushita T,Rama A,Charolidi N,et al.Relationship of connexin43 expression to phenotypic modulation in cultured human aortic smooth muscle cells[J].Eur J Cell Biol,2007,86(10):617-28.
[11]Johnstone S R,Kroncke B M,Straub A C,et al.MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation[J].Circ Res,2012,111(2):201-11.
[12]Song M,Yu X,Cui X,et al.Blockade of connexin 43 hemichannels reduces neointima formation after vascular injury by inhibiting proliferation and phenotypic modulation of smooth muscle cells[J].Exp Biol Med(Maywood,NJ),2009,234(10):1192-200.
[13]Kwak B R,Veillard N,Pelli G,et al.Reduced connexin43 expression inhibits atherosclerotic lesion formation in low-density lipoprotein receptor-deficient mice[J].Circulation,2003,107(7):1033-9.
[14]Shi Y,Hou X,Zhang X,et al.Inhibition of oxidized-phospholipid-induced vascular smooth muscle cell proliferation by resveratrol is associated with reducing Cx43 phosphorylation[J].J Agric Food Chem,2013,61(44):10534-41.
[15]侯晓敏,秦小江.槲皮素通过激活kv1.5保护糖尿病大鼠冠脉损伤[J].中国药理学通报,2017,33(10):1442-5.[15]Hou X M,Qin X J.Quercetin protects coronary artery from injury induced by diabetes in rats by activating Kvl.5[J].Chin Pharmacol Bull,2017,33(10):1442-5.
[16]Liu Y,Fu Y Q,Peng W J,et al.Rutaecarpine reverses the altered connexin expression pattern induced by oxidized low-density lipoprotein in monocytes[J].J Cardiovasc Pharmacol,2016,67(6):519-25.